Quantitative analysis of cells and analytes in fluid samples, particularly bodily fluid samples, often provides critical diagnostic and treatment information for physicians and patients. Quantitative immunoassays utilize the specificity of the antigen (Ag)-antibody (Ab) reaction to detect and quantitate the amount of an Ag or Ab in a sample. In solid phase immunoassays, one reagent (e.g., the Ag or Ab) is immobilized to a solid surface, facilitating separation of bound reagents or analytes from free reagents or analytes. The solid phase is exposed to a sample containing the analyte, which binds to its Ag or Ab; the extent of this binding is quantitated to provide a measure of the analyte concentration in the sample. Transduction of the binding event into a measurable signal, however, is affected by a number of interferences, such as variability in binding of components of the assay, which are not associated with the presence or amount of the analyte. These interferences limit the specificity and applicability of quantitative immunoassays.
Over the years, numerous simplified test systems have been designed to rapidly detect the presence of a target analytes of interest in biological, environmental and industrial fluids. In one of their simplest forms, these assay systems and devices usually involve the combination of a test reagent which is capable of reacting with the target analytes to give a visual response and an absorbent paper or membrane through which the test reagents flow. Paper products, glass fibers any nylon are commonly used for the absorbant materials of the devices. In certain cases, the portion of the absorbent member containing the test reagents is brought into contact, either physically or through capillarity, with the sample containing the target analytes. The contact may be accomplished in a variety of ways. Most commonly, an aqueous sample is allowed to traverse a porous or absorbent member, such as porous polyethylene or polypropylene or membranes by capillarity through the portion of the porous or absorbent member containing the test reagents. In other cases, the test reagents are pre-mixed outside the test device and then added to the absorbent member of the device to ultimately generate a signal.
With the advancement of microfluidics, it is possible that these diagnostic devices are made in a compact form to facilitate the immunoassays testing. In U.S. Pat. No. 6,905,882, it is reported the assay devices, assay systems and device components comprise at least two opposing surfaces disposed a capillary distance apart, at least one of which is capable of immobilizing at least one target analyte or a conjugate in an amount related to the presence or amount of target analyte in the sample from a fluid sample in a zone for controlled fluid movement to, through or away the zone. The device components may be incorporated into conventional assay devices with membranes or may be used in the inventive membrane-less devices herein described and claimed. These components include flow control elements, measurement elements, time gates, elements for the elimination of pipetting steps, and generally, elements for the controlled flow, timing, delivery, incubation, separation, washing and other steps of the assay process.
Methods for quantitatively measuring the amount of an analyte of interest in a fluid sample are disclosed in US Pub. No. 2004/0171092. The methods involve providing a membrane having an application point, a contact region comprising analyte-binding particles, a sample capture zone, and a control capture zone, where the contact region is between the application point and the sample capture zone, and the sample capture region is between the contact region and the control capture zone. In the assays, a fluid allows transport components of the assay by capillary action through the contact region, to and through the sample capture zone and subsequently to and through the control capture zone. In a “sandwich assay” embodiment, the amount of analyte in the fluid sample is related to a corrected analyte-binding particle amount, which can be determined, for example, as a ratio of the amount of analyte-binding particles in the sample capture zone and the amount of analyte-binding particles in the control capture zone. In a “competitive assay” embodiment, the membrane has an application point, a contact region comprising analyte-coated particles, a sample capture zone, and a control capture zone, where the contact region is between the application point and the sample capture zone, and the sample capture zone is between the contact region and the control capture zone. In this “competitive assay” embodiment, the amount of analyte in the fluid sample is inversely related to a corrected analyte-coated particle amount, which can be determined, for example, as a ratio of the amount of analyte-coated particles in the sample capture zone and the amount of analyte-coated particles in the control capture zone.
Active devices for immunoassays are also reported in U.S. Pat. No. 6,887,362. The report involves devices and methods for performing active, multi-step molecular and biological sample preparation and diagnostic analyses employing immunochemical techniques. It relates generally to bioparticle separation, bioparticle enrichment, and electric field-mediated immunochemical detection on active electronic matrix devices utilizing AC and DC electric fields. More specifically, the invention relates to devices and methods for sample preparation/manipulation, immunoimmobilization, and immunoassays, all of which can be conducted on one or more active electronic chip devices within a single system. These manipulations are useful in a variety of applications, including, for example, detection of pathogenic bacteria and biological warfare agents, point-of-care diagnostics, food or medical product quality control assays, and other biological assays.
Traditional immunoassay methods utilizing microtiter-plate formats, dipsticks, etc., are labor and time extensive. Multiple steps requiring human intervention either during the process or between processes are sub-optimal in that there is a possibility of contamination and operator error. Further, the use of multiple machines or complicated robotic systems for performing the individual processes is often prohibitive except for the largest laboratories, both in terms of the expense and physical space requirements.
The microfluidics based devices reported in U.S. Pat. No. 6,905,882 and US Pub. No. 2004/0171092 eliminated most parts of human intervention. However, the key parameters in the immunoassays process, such as the incubation time, the flow of fluid, and the mixing of fluorescent tags with detection antibody are accomplished with the passive capillary forces. The passive approach of moving fluid in microfluidics relies on the capillary force. Because each type of fluid has its own viscosity, and the fluid viscosity is also temperature dependant, the amount of fluid that flows in the microfluidics is hence not accurately controlled in these passive devices. The active device reported in the U.S. Pat. No. 6,887,362 relies on the electric field to move and analyze the analytes. This approach suffers from the limitation that analytes will have to be charged particles to be analyzed in the said devices.